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Gunasekera, S., Muhammad, T., Strömstedt, A. A., Rosengren, K. J. & Göransson, U. (2018). Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity. ChemBioChem (Print), 19(9), 931-939
Open this publication in new window or tab >>Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 9, p. 931-939Article in journal (Refereed) Published
Abstract [en]

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
antibiotics, cytotoxicity, drug discovery, peptides, structure-activity relationships
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356391 (URN)10.1002/cbic.201700599 (DOI)000431625100008 ()29430821 (PubMedID)
Funder
Swedish Research Council, 2011-3403Carl Tryggers foundation , CTS 10: 126Carl Tryggers foundation , CTS 11: 169Swedish Society of Medicine, SLS-254511
Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2018-07-25Bibliographically approved
Slazak, B., Kapusta, M., Strömstedt, A. A., Slomka, A., Krychowiak, M., Shariatgorji, M., . . . Göransson, U. (2018). How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System. Frontiers in Plant Science, 9, Article ID 1296.
Open this publication in new window or tab >>How Does the Sweet Violet (Viola odorata L.) Fight Pathogens and Pests - Cyclotides as a Comprehensive Plant Host Defense System
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2018 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, article id 1296Article in journal (Refereed) Published
Abstract [en]

Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet (Viola odorata L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens (Fusarium oxysporum, F. graminearum, F. culmorum, Mycosphaerella fragariae, Botrytis cinerea) and fungi isolated from violets (Colletotrichum utrechtense and Alternaria alternata), with minimal inhibitory concentrations (MICs) ranging from 0.8 to 25 mu M. Inhibition of phytopathogenic bacteria - Pseudomonas syringae pv. syringae, Dickeya dadantii and Pectobacterium atrosepticum - is also observed with MIC = 25-100 mu M. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest ( ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in V. odorata, in which different types of peptides have specific targets that determine their distribution in plant tissues.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2018
Keywords
cyclotides, plant host defense, Violaceae, antimicrobial peptide, antifungal defense, MALDI-MSI, immunohistochemistry
National Category
Botany
Identifiers
urn:nbn:se:uu:diva-365299 (URN)10.3389/fpls.2018.01296 (DOI)000444243600001 ()30254654 (PubMedID)
Funder
Swedish Research Council, 621-2007-5167Swedish Research Council, 621-2014-6215Swedish Foundation for Strategic Research , RIF14-0078Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-11-13 Created: 2018-11-13 Last updated: 2018-11-13Bibliographically approved
Jacobsson, E., Andersson, H. S., Strand, M., Peigneur, S., Eriksson, C., Lodén, H., . . . Göransson, U. (2018). Peptide ion channel toxins from the bootlace worm, the longest animal on Earth. Scientific Reports, 8, Article ID 4596.
Open this publication in new window or tab >>Peptide ion channel toxins from the bootlace worm, the longest animal on Earth
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4596Article in journal (Refereed) Published
Abstract [en]

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the alpha-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long alpha-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight alpha-nemertides, mainly distributed in the genus Lineus. alpha-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 mu g/kg (similar to 300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Na(v)1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that a-nemertides can be promising candidates for development of bioinsecticidal agents.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-351585 (URN)10.1038/s41598-018-22305-w (DOI)000428029600001 ()29567943 (PubMedID)
Funder
Swedish Research Council, 2014-3327]
Available from: 2018-05-29 Created: 2018-05-29 Last updated: 2018-05-29Bibliographically approved
Wang, Y., Lloyd, K. A., Gunasekera, S., Eriksson, C., Ramsköld, D., Lundberg, K., . . . Grönwall, C. (2018). Repertoire Studies in Rheumatoid Arthritis Reveal B-Cell Distortions and Baseline Shifts in Unmutated IgG. Arthritis & Rheumatology, 70
Open this publication in new window or tab >>Repertoire Studies in Rheumatoid Arthritis Reveal B-Cell Distortions and Baseline Shifts in Unmutated IgG
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2018 (English)In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 70Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
John Wiley & Sons, 2018
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:uu:diva-369631 (URN)000447268900009 ()
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2018-12-17Bibliographically approved
Gunasekera, S., Fernandes-Cerqueira, C., Wennmalm, S., Wahamaa, H., Sommarin, Y., Catrina, A. I., . . . Göransson, U. (2018). Stabilized Cyclic Peptides as Scavengers of Autoantibodies: Neutralization of Anticitrullinated Protein/Peptide Antibodies in Rheumatoid Arthritis. ACS Chemical Biology, 13(6), 1525-1535
Open this publication in new window or tab >>Stabilized Cyclic Peptides as Scavengers of Autoantibodies: Neutralization of Anticitrullinated Protein/Peptide Antibodies in Rheumatoid Arthritis
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2018 (English)In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 13, no 6, p. 1525-1535Article in journal (Refereed) Published
Abstract [en]

The occurrence of autoantibodies is a hallmark of rheumatoid arthritis, specifically those autoantibodies targeting proteins containing the arginine-derived amino acid citrulline. There is strong evidence showing that the occurrence of anticitrullinated protein/peptide antibodies (ACPA) are involved in disease progression, and ACPA was recently shown to induce pain in animals. Here, we explore a novel concept useful for research, diagnostics, and possibly therapy of autoimmune diseases, namely, to directly target and neutralize autoantibodies using peptide binders. A high-affinity peptide-based scavenger of ACPA was developed by grafting a citrullinated epitope derived from human fibrinogen into a naturally occurring stable peptide scaffold. The best scavenger comprises the truncated epitope alpha-fibrinogen, [Cit573]fib(566-580), grafted into the scaffold sunflower trypsin inhibitor-1, SFTI-1. The final peptide demonstrates low nanomolar apparent affinity and superior stability.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:uu:diva-358527 (URN)10.1021/acschembio.8b00118 (DOI)000435746200015 ()29630823 (PubMedID)
Funder
Swedish Research Council, 2012-5063Swedish Research Council, 2017-02577Swedish Foundation for Strategic Research , F06-0058Swedish Rheumatism Association, R-755861Stockholm County Council, 20160378
Available from: 2018-09-03 Created: 2018-09-03 Last updated: 2018-09-03Bibliographically approved
Strömstedt, A. A., Park, S., Burman, R. & Göransson, U. (2017). Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra. Biochimica et Biophysica Acta - Biomembranes, 1859(10), 1986-2000
Open this publication in new window or tab >>Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra
2017 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, no 10, p. 1986-2000Article in journal (Refereed) Published
Abstract [en]

Cyclotides are a family of plant peptides characterized by a cystine knot embedded in a macrocyclic backbone. They bind to and disrupt phospholipid membranes, which explain their lytic activity on cells. In this study, we expose the full antibacterial potency of cyclotides by avoiding its inhibition by rich growth media assay conditions. For that purpose a two-step microdilution assay protocol was developed, using non-growing conditions during initial peptide incubation. A diverse set of cyclotides was tested for antibacterial and antifungal activity, and the results show that most cyclotides are active under these conditions, especially against Gram-negative bacteria. Activity was observed at sub-micromolar concentrations for three of the cyclotides tested, surpassing that of the control peptides LL-37 and melittin. Noteworthy, two anionic cyclotides were active on Pseudomonas aeruginosa at low micromolar concentrations. Broad-spectrum activity was pronounced among cycloviolacin cyclotides, which included activity on Staphylococcus aureus and Candida albicans. The factors influencing their bactericidal spectrum were revealed by correlating antimicrobial activity with membrane permeabilization on various liposome systems and with the physiochemical properties of the cyclotides. Whereas general electrostatic and hydrophobic parameters are more important for broad-spectrum cyclotides; a phospholipid-specific mechanism of membrane permeabilization, through interaction with phosphatidylethanolamine-lipids, is essential for cyclotides active primarily on Gram-negative bacteria.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2017
Keywords
Cyclotide, Antimicrobial peptide, Antibacterial, Membrane permeabilization, Phosphatidylethanolamine-binding, Structure-activity relationship
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-336446 (URN)10.1016/j.bbamem.2017.06.018 (DOI)000411419000024 ()28669767 (PubMedID)
Funder
Swedish Society of Medicine, SLS-254511Swedish Research Council, 2012-5063
Available from: 2017-12-14 Created: 2017-12-14 Last updated: 2017-12-14Bibliographically approved
Hellinger, R., Thell, K., Vasileva, M., Muhammad, T., Gunasekera, S., Kuemmel, D., . . . Gruber, C. W. (2017). Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins. Frontiers in Chemistry, 5, Article ID 73.
Open this publication in new window or tab >>Chemical Proteomics for Target Discovery of Head-to-Tail Cyclized Mini-Proteins
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2017 (English)In: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 5, article id 73Article in journal (Refereed) Published
Abstract [en]

Target deconvolution is one of the most challenging tasks in drug discovery, but a key step in drug development. In contrast to small molecules, there is a lack of validated and robust methodologies for target elucidation of peptides. In particular, it is difficult to apply these methods to cyclic and cysteine-stabilized peptides since they exhibit reduced amenability to chemical modification and affinity capture; however, such ribosomally synthesized and post-translationally modified peptide natural products are rich sources of promising drug candidates. For example, plant-derived circular peptides called cyclotides have recently attracted much attention due to their immunosuppressive effects and oral activity in the treatment of multiple sclerosis in mice, but their molecular target has hitherto not been reported. In this study, a chemical proteomics approach using photo-affinity crosslinking was developed to determine a target for the circular peptide [T20K]kalata B1. Using this prototypic nature-derived peptide enabled the identification of a possible functional modulation of 14-3-3 proteins. This biochemical interaction was validated via competition pull down assays as well as a cellular reporter assay indicating an effect on 14-3-3-dependent transcriptional activity. As proof of concept, the presented approach may be applicable for target elucidation of various cyclic peptides and mini-proteins, in particular cyclotides, which represent a promising class of molecules in drug discovery and development.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
cyclotides, cyclic protein, chemical proteomics, peptide-protein interaction, photo-affinity labeling
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-338516 (URN)10.3389/fchem.2017.00073 (DOI)000412726000001 ()29075625 (PubMedID)
Funder
Australian Research Council, FT140100730
Available from: 2018-01-15 Created: 2018-01-15 Last updated: 2018-01-15Bibliographically approved
Park, S., Yoo, K.-O., Marcussen, T., Backlund, A., Jacobsson, E., Rosengren, K. J., . . . Göransson, U. (2017). Cyclotide Evolution: Insights from the Analyses of Their Precursor Sequences, Structures and Distribution in Violets (Viola). Frontiers in Plant Science, 8, Article ID 2058.
Open this publication in new window or tab >>Cyclotide Evolution: Insights from the Analyses of Their Precursor Sequences, Structures and Distribution in Violets (Viola)
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2017 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 8, article id 2058Article in journal (Refereed) Published
Abstract [en]

Cyclotides are a family of plant proteins that are characterized by a cyclic backbone and a knotted disulfide topology. Their cyclic cystine knot (CCK) motif makes them exceptionally resistant to thermal, chemical, and enzymatic degradation. By disrupting cell membranes, the cyclotides function as host defense peptides by exhibiting insecticidal, anthelmintic, antifouling, and molluscicidal activities. In this work, we provide the first insight into the evolution of this family of plant proteins by studying the Violaceae, in particular species of the genus Viola. We discovered 157 novel precursor sequences by the transcriptomic analysis of six Viola species: V. albida var. takahashii, V. mandshurica, V. orientalis, V. verecunda, V. acuminata, and V. canadensis. By combining these precursor sequences with the phylogenetic classification of Viola, we infer the distribution of cyclotides across 63% of the species in the genus (i.e., ~380 species). Using full precursor sequences from transcriptomes, we show an evolutionary link to the structural diversity of the cyclotides, and further classify the cyclotides by sequence signatures from the non-cyclotide domain. Also, transcriptomes were compared to cyclotide expression on a peptide level determined using liquid chromatography-mass spectrometry. Furthermore, the novel cyclotides discovered were associated with the emergence of new biological functions.

Keywords
cyclotide evolution, viola phylogeny, sequence signature, cyclotide precursor, neofunctionality, novel cyclotide, precursor domain
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:uu:diva-339782 (URN)10.3389/fpls.2017.02058 (DOI)000418117800001 ()29326730 (PubMedID)
Funder
Swedish Research Council, 2012-5063
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-02-09Bibliographically approved
Malik, S. Z., Linkevicius, M., Göransson, U. & Andersson, D. I. (2017). Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides. Antimicrobial Agents and Chemotherapy, 61(8), Article ID e00684-17.
Open this publication in new window or tab >>Resistance to the Cyclotide Cycloviolacin O2 in Salmonella enterica Caused by Different Mutations That Often Confer Cross-Resistance or Collateral Sensitivity to Other Antimicrobial Peptides
2017 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 61, no 8, article id e00684-17Article in journal (Refereed) Published
Abstract [en]

Antimicrobial peptides (AMPs) are essential components of innate immunity in all living organisms, and these potent broad-spectrum antimicrobials have inspired several antibacterial development programs in the past 2 decades. In this study, the development of resistance to the Gram-negative bacterium-specific peptide cycloviolacin O2 (cyO2), a member of the cyclotide family of plant miniproteins, was characterized in Salmonella enterica serovar Typhimurium LT2. Mutants isolated from serial passaging experiments in increasing concentrations of cyO2 were characterized by whole-genome sequencing. The identified mutations were genetically reconstituted in a wild-type background. The additive effect of mutations was studied in double mutants. Fitness costs, levels of resistance, and cross-resistance to another cyclotide, other peptide and nonpeptide antibiotics, and AMPs were determined. A variety of resistance mutations were identified. Some of these reduced fitness and others had no effect on fitness in vitro, in the absence of cyO2. In mouse competition experiments, four of the cyO2-resistant mutants showed a significant fitness advantage, whereas the effects of the mutations in the others appeared to be neutral. The level of resistance was increased by combining several individual resistance mutations. Several cases of cross-resistance and collateral sensitivity between cyclotides, other AMPs, and antibiotics were identified. These results show that resistance to cyclotides can evolve via several different types of mutations with only minor fitness costs and that these mutations often affect resistance to other AMPs.

Keywords
antimicrobial peptide resistance, cyclotide, cycloviolacin O2, cross-resistance, collateral sensitivity, Salmonella enterica, antimicrobial peptides, fitness, mechanisms of resistance, Salmonella enterica serovar Typhimurium
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-333077 (URN)10.1128/AAC.00684-17 (DOI)000406259400067 ()
Funder
Swedish Research Council
Available from: 2017-11-06 Created: 2017-11-06 Last updated: 2018-01-13Bibliographically approved
Uddin, S. J., Muhammad, T., Shafiullah, M., Slazak, B., Rouf, R. & Göransson, U. (2017). Single-step purification of cyclotides using affinity chromatography. Biopolymers, 108(3), Article ID e23010.
Open this publication in new window or tab >>Single-step purification of cyclotides using affinity chromatography
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2017 (English)In: Biopolymers, ISSN 0006-3525, E-ISSN 1097-0282, Vol. 108, no 3, article id e23010Article in journal (Refereed) Published
Abstract [en]

Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Mobius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

Keywords
affinity chromatography, cyclotides, cycloviolacin O2, immobilized IgG
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:uu:diva-334107 (URN)10.1002/bip.23010 (DOI)000407992300005 ()
Funder
Swedish Research Council, 2012-5063
Available from: 2017-11-24 Created: 2017-11-24 Last updated: 2017-11-24Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-5005-9612

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